MIDWEST RESEARCH INSTITUTE

SMAMMALIAN TOXICITY OF MUNITIONS COePOUNDS PHASE ACUTE ORAL

I::

TOXICITY, PRIMARY SKIN AND EYE IRRITATION, DERMALSENSITIZATION, DISPOSITION AND METABOLISM,

AND AMES TESTS OF ADDITIONAL COMPOUNDS

CPROGRESS REPORT NO. 6December 8, 1978 . .

Contract No. DAMD-17-74-C-4073MRI Project No. 3900-B . ..-- --

DiSTWrA1BO71N STATEMEN4T ACID_ Approved for publici release;

For DistributLon Unlimited

[ Contract Officer's Technical Representative: Dr. Jack C. DacreEnvironmental Protection Research Division

U.S. Army Medical Bioengineering Research andDevelopment Laboratory

Fort Detrick, Frederick, Maryland 21701

M I60

MIDWEST RESEARCH INSTITUTE 425 VOLKER BOULEVARD, KANSAS CITY, MISSOURI 64110 *816 753-7600

S............ . +,•e,. +,_.,.. " - -- ,,ht.qL + "

.1

21Animal Experimentation: Animal experiments were conducted according toT'he "Gtkide for the Care and Use of Laboratory Animals" (1974) preparedby the Institute of Laboratory. Animal Resources, National Research Council;the regulations and standards prepared by the Department of Agriculture;and Public Law 91-570, "Laboratory Animal Welfare Act," 1970.

Disclaimer: The findings in this report are not to be construed as an iiofficial Der~rtment of the Amy position unless so designated by otherauthorized documents.

,.1

I:I

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MAMMALIAN TOXICITY OF MUNITIONS COMPOUNDS PHA&E I: ACUTE ORALTOXICITY, PRIMARY SKIN AND EYE IRRITATION, DERMAL

F SENSITIZATION, DISPOSI1 ON AND METABOLISM,AND AMES TESTS OF ADDLITONAL COMPOUNDS

PROCRESS REPORT NO. 6<I December 8, 1978

K1 ByHarry V. Ellis, III

John R. Hodgson

Ii Shang W. Hwang

Laurel M. HalxpapDanny 0. Helton

Bruce S. AndersenDaniel L. VanGoethem

|- Cheng-Chun Lee

Supported by

U.S. Army Medical Research and Development CommandFort Detrick, Frederick, Maryland 21701

Contract No. DAMD-17-74-C-4073

[ MRI Project No. 3900-B

[ Contract Officer's Technical Representative: Dr. Jack C. Dacre

Environmental Protection Research Division

U.S. Army Medical Bioengineering Research and

Development LaboratoryFort Detrick, Frederick, Maryland 21701

Distribution Unlimited

ItRJ (OUWN STA'1 u~pr AkAppr•,mv'i f,.. pubI w IL J, Os

MIDWEST RESEARCH INSTITUTE 425 VOLKER BOULEVARD, KANSAS CITI, MISSOURI 64110 816 753-7600

SCCURITY CLASSIFICATION OF THIS PAGs ("O~n Dats, Entore.REPORT DOCUMENTATION PAGE RA NTUrm

I~WWhT UMUE I~GOVT ACCESSION4 NO. 2. RECIPIRNT S CATALOG NUMBERt

Progress Report No. 6 /I_______________[. C. TYPE -,F RYPORT a PERIOD COVERED

Mammalian Toxicity of munitions compounds.Phase 11 Progress, epert-[Acute Oral Toxicity, Primary Skin and Eye Irrita- Feb. 1. 1975 - Feb._28, 1978tion, Dermal Sensitization, Disposition and Me- *- PERFORMING OR4.REPORTMU till

I ~ ~~rruce S. Andersen, Daniel I >iu",tnoethýein, eDAf7-4C43

S. PERFORMING ORGANIZATION NAME AND IDRESS

I I. CONTI, ILLING OFFICE NAME AND ADDRESS

For~t Detrick, Frederick, Maryland 21701

ItMONITORING AGENCY NAME & AOORESSQif different from Contr'olling Office) IS. SECURITY CLASS. (of thisE report)

U.S. Army Medical Bioengineering Research andDevelopment Laboratory Unclassified

Fort Detrick, Frederick, Maryland 21701 150. DEL SSI.IC ATION/ DOWNGRADING

IS. DISTRIBUTION STATEMENT (of this steport

I: ~Distribution unlimited

17 IST R11UTIOI4 str AlEME,4T (of the abetraed entered In Block 20, it different frogn Report)

coIiP es- ýIs. SUPPLEMENTARY NOTES

1.KEY WORDS (Continuo an revere* oid* It necessary and identify by block numnber)Trinitrotoluene (TNT; 2-Methyl-l,3,5-trinitrobenzene, CAS Reg. No. 118-96-7)2,3-Dinitrotoluene (I-Methyl-2,3--dinitrobenzene, CAS Reg. No. 602-01-7)2,4-Dini'ý.:otoluene (1-Methyl-2,4-dinitrobenzene, CAS Reg. No. 121-14-2)2,5-Dinitrotoluene (2-Methyl-1,4-dinitrobenzene, CAS Reg. No. 619-15-8)2.6-Dinitrotoluene (2-Methvl-1.3-dinitrobenzene, CAS Reg. No. 606-20-2)_

20. ABSTRACT (Continue an reverse. ode If nec~esary mud Identify by block umber)

This report supplements Progress Report No. 1, dated July 22, 1975.)23',5-DiMnitrotoluene (3,5-DNT) was the most potent of all DNT isomers in oral acut3doses to rats and mice. 2-Amino-4,6-DNT (2-ADNT) and its isomer, 4-ADNTT, werethe least Votent in rats Lnd female mice, and comparable to 2,3-DNT and 2,4-DNTin male mice. 3,5-DNT anc' 4-ADNT were not irritating to rabbit skin; 2-ADNTwas a mild irritant. All three compounds w'ete not irritating to rabbit eyesand not sensitizing to guinea pigs. t.rDD 1473 EDITION 0OF 1 NOV 66 1S O¶4SOL!tE

SECURITY CLASSIFICATION OF THIS PAGE9 (When Does Enitered)I: w

LUI*TY OLAUUPSCATION O THIS t!2l6(ff Soft"

A (concluded)

ý,4-Dinitrotoluene (4-Methyl-3.,2-dinitrobenzene, CAS Reg. No. 610-39-9)1,5-Dinitrotoluene (1-Methyl-5,5-dinitrobenzene, CAS Reg. No. 618-85-9)

j1-Amino-4 ,6-dinitrotoluene (2-Methyl-3, 5-dinitroberizenamine * CAS Reg. No.35572-78-2)

I-Amino-2,6-dinitrotoluene (4-?4ethyl-3, 5-dinitrobenzenamine, GAS Reg. No.19406-51-0)

letranitromethane (CAS Reg. No. 509-14-8)ýrinitroglycerin (1,2,3-Propanetriol trinitrate, GAS Reg. No. 55-63-0)ý.,2-Dinitroglycerin (1,2,3-Propanetriol 1,2"-dinitrate, GAS Reg, No. 621-65-8)h.,3-Diaitroglycerin (1,2,3-Propanetriol 1,3-dinitrate, CAS Reg. No. 623-87-0)h.-Mononitroglycerin (1,2.3-Propanetri~ol 1-nitrate, CAS Reg. No. 624-43-1)I-.Mononitroglycerin (1,2,3-Propanetriol 2-nitrate, CAS Reg. No. 620-12-2)itrocellulose (Cellulose nitrate, CAS Reg. No. 9004-70-0)ite Phosphorus (CAS Reg. No. 12185-10-3) -

cute Toxicity1.spositiontabolismes test

0 (concluded)

,5-DNT and 4-ADNT were absorbed~ from the gastrointestinal tract, metabolizednd excreted in the urine.ni the Am~es test, 1,3-dinitroglycerin (1,3-DNG), 1-mononitroglycerin (1-MNG),itrocellulose and white phosphorus were not mutagenic. Trinitrotoluene (TNT) -

,4-DNT, 2,5-DNT, tetranitromethane (TNM) arnd l,2-DNG were mutagenic at 10 too jig/plate in one or more strains. TNM was bactericidal without activation.,2-DNG was nonmutagenic with activation. 2,3-DNT, 2,6-DNT, 3,5-DNT, trinitro-lycerin and 2-MNG were weakly mutagenic, with mutagenic results at 100 or -

,000 jig/plate in one or more strains.

IRCUMIY CLASSIFICATION OF THIS PAGE(Whwm bae ta 5n1.4)

PREFACE

This report was prepared at Midwest Research Institute, 425

Volker Boulevard, Kansas City, Missouri 64110, under U.S. Department ofthe Army, Contract No. 073, MRI Project No. 3900-B, "MunitionsCompounds Mammallin Toxicity Study." The rwa s_ s-uppor-ted-- the Medi-cal Research and Development Command, Department of the Army.Captain John P. Glennon and Dr. Jack C. Dacre, Environmental Protection

Research Division, USAMBRDL, were the successive contract officer'stechnical representatives for the project.

This work was conducted in the Biological Sciences Divisionunder the direction of Dr. William B. Houae, between February 1, 1975

and February 28, 1978. The experimental work was directed by-r. Cheng-CEhun-tee, 1iucipal Advisor for Pharmacology/Toxicology, with

the assistance of Dr. Harry V. r'lis, III, Senior Pharmacologist.Dr. John R. AIodgson, Head, Biochemical and Developmental Pharmacology,supervised the studies on disposition and metabolism, assisted byDr. Shang W. Hwang and Miss Laurel M. Halpap, and the Ames tests, as-sited by Mr. Daniel L. VanGCeethem. Dr. Danny 0. Helton, Senior Chemist,prepared the 3,5-dinitrotoluene. Mr. Bruce S. Andersen assisted withthe in vivo toxicity studies.

Approved for:

MIDWEST RESEARCH INSTITUTE

CheikCh~LeePrincipal Advisor forPharmacology/Toxicology

December 8, 1978

A\- I

vii Iu /~t orD! p~ta

TABLE OF CON4TENTS

Page

I. Introduction. . . . . ........ . . . . . . . . . . 1

II. Materials and Methods ...... .............. . . . . . 3

A. Animals ......... ....................... 3B. Chemicals ............... .................... 3C. Acute LD 5 0 . . . . .. .. .. .. .. . . . . . . . . . .. . . . . . . . . 4D. Primary Skin and Eye Irritation. ......... 4E. De-.mal Sensitivity Studies . .......... 4F. Disposition and Metabolism ...... ........... 4

1. Radiochemicals ......... ............... 42. Experimental Procedures . . . . . . . . . . 43. Rad:'oactive Assays .... ............... . 5

4. Thin--Layer Chromatography (TLC) ... ...... 5G. Ames Test ............... ................... 5

1. Preparation of Test Compounds ........ 5

2. Bacteria and Culture Media ..... ......... 63 Preparation of Rat Liver S-9 Fraction . . . 64. Microjomal Activation System .... ....... 65. Assay Procedure .Atai ................. 7

III. Results .......... ..... ......................... 9

A. Synthesis of 3,5-DNT ... i................ 9

A . Identity . .. ...................... 9

a. Capillary Melting Point. ........ 9

b. Infrared Spectrum ..... ........... 9c. Nuclear Magnetic Resonance .... ...... 9

2. Assay .............. ................... 9

a. Gas Chromatography (GC) ......... . 9b. Thin-Layer Chromatography (TLC) . ... 10

c. Discussion .... .............. ... 10

B. In Vivo Studies ...... .................. 10

1. 3,5-Dinitrotoluene ....... ............. 102. 2-Amino-4,6-Dinitrotoluene ........ . . . . 11

ix

TABLE OF CONTENTS (continued)

page

3. 4-Amino-2,6-Dinitrotoluene. . . . . . . . 11

C. Ames Tests .......... ................... ... 12

IV. Discussion ....... ....................... . . . 27

A. Acute Oral Toxicity in Rats .............. .... 27

B. Acute Oi'al Toxicity in Mice .............. .... 27C. Toxic Effects ....... ................ . 27D. Primary Skin and Eye Irritation ........... ... 28E. Dermal Sensitivity ...... ............... ... 28F. Disposition and Metabolism ... ........... .... 28

G. Ames Tests .............. ................... 28

V. Conclusions ............ ....................... .. 31

V1. Corrigenda to Report No. 1 ...... ................... 33

References............... ................. ................. . 35

List of Figures -,

Figure Title Page

1 TLC of Rat Urine After Oral Administration of 3,5-DNT(Ring-UL-1 4 C) ............ .................... . 25

2 TLC of Rat Urine After Oral Administration of 4-ADNT(Ring-UL-1 4C) .......... ..................... .... 26

List of Tables

Table Title Page

1 Acute Oral Toxicities (mg/kg) of Various NitrotolueneCompounds in Male and Female Rats ... ........... .... 14

2 Acute Oral Toxicities (mg/kg) of Various NitrotolueneCompounds in Male and Female Mice ... ........... .... 15

x

List of Tables (concluded)

Table Title Page

3 Primary Skin Irritation of Various Nitrotoluene Com-pounds in Rabbits. . . ................ . . . 16

4 Primary Eye Irritation of Various Nitrotoluene Com-pounds in Rabbits. ........................ . 17

5 Dermal Sensitivity of Various Nitrotoluene Compounds inGuinea Pigs ....... ....................... 18

6 Distribution and Excretion of Radioactivity in Rats he-- aeiving 3,5-DNT-(Rig-tL-l 4 C). ............... 19

7 Distribution and Excretion of Radioactivity in Rats Re-ceiving 4-Amino-2,6-Dinitrotoluene-(Ring-UL-1 4 C) . . . 20

8 Mutagenicity of Munitions Compounds Using the Salmonella/Microsome Plate Test . .. .................. 21

9 Summary of lutagenicity of Munitions Compounds Using theSalmonella/Microsome Plate Test. . . . . . . . . . . . 24

1x.Ii1. 4

1.

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1.• I. INTRODUCTION

Unuer Contract No. DAND-17-74-C-4073, entitled "Muinitions CompoundsHammalian Toxicity Study," we have completed various studies in Phase I.Most results were reported in Progress Report No. 1, dated July 22, 1975.1/

tI Because sufficient compound waa not yet available, several compounds were

not tested until after that report was complet3d. This report includes

the acute oral toxicity in rats and mice, primary skin and eye irritationj in rabbits and dermal sensitivity in guinea pigs of 3,5-dinitrotoluene

(3,5-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), and 4-amino-2.6-dinitro-

toluene (4-ADNT), and the disposition and metabolism of 1 4 C-labeled 3,5-DNT

and 4-ADNT after oral administration to rats.

The so-called "Ames Test" 2 a_ 2 b/ is becoming more popular as arapid screen for mutagenicity. Therefore, we have performed the test onavailable compounds, including tetranitromethane (TNM), 2,4,6-trinitrotol..

irene (TNT), 2,3-dinitrotoluetne (2,3-DNT), 2,4-dinitrotoluene (2,4-DNT),2,5-dinitrotoluene (2,5-DNT), 2,6,-.dinitrotoluene (2,6-DNT), 3,4-dinitro-toluene (3,'.-DNT), 3,5-DNT, trinitroglycerin (TNG), 1,2-dinitroglycerin

(l,2-DNG), 1,3-dinttroglycerin (l,3-DNG), 1-mononitroglycerin (1-141N),

2-mononitroglycerin (2-MNG), nitrocellulose (NC), and white phosphorus(WP).

A corrigenda for Report No. 1 is also included.

Results of studies on the absorption and dispositon of 1 4 C-labeled

NC were included in Report No. 5.3/ Further chemical analysis of NC fines

was reported separately.4/

hi1"-:- |a.

II. MATERIALS AND METHODS

A. Animals

Male and female Charles River CDOrats were obtained from theCharles River Breeding Laboratories (North Wilmington, Massachusetts).Male and female albino Swiss mice werE obtained from the NationalLaboratory Animal Company (O'Fallon. Missouri). Guinea pigs and NewZealand rabbits were obtained from Small Stock Industries (Pea Ridge,Arkansas).

All animals were kept in air-conditioned rooms (75 + 5*F) withrelative humidity of 50 + 10% and photoperiod of 12 hr. They were keptunder observation for 1 week after arrival; only healthy animals wereused. Rats, mice, Lnd guinea pigs were housed in plastic cages and pro-vided with hardwood bedding; rabbits were housed in metal rages with wirebottoms.

B. Chemicals

3,5-DNT was prepared at Midwest Research Inatitute (HRI) byDr. Danny 0. Helton, according to the method of Morton and McCookin,.!/as outlined below. 2-ADNT and 4-ADNT were obtained from the lateDr. Lloyd A. Kaplan of the Naval Surface Weapons Center, White Oak(Silver Spring, Maryland). TNM was purchased from Aldrich ChemicalCompany, Milwaukee, Wisconsin. All other chemicals wert those usedearlier.l/

CH3 CH3 4H 3 H

S Ac 20 HNOI HSO4

0N'N202N 02

N2 HNCOCH3 HNCOCH 3 N 2

(1) HCl, NaNO 2

(2) H3P0 2

CH3

02 & 02

3

...........

C. Acute LD 5 0

Rate and mice were fasted for at least 16 hr prior to oral dosing.Oral doses of 5% suspensions in peanut oil were administered to the rodentsby intragastirc istubation via a stainless steel tube. After treatment,the survivors were observed daily for 14 days for delayed mortality ortoxic signs. The LD5 0 was calculated for each compound by a computerprogram based on the maximum likelihood method of Finney.A/

D. Primary Skin and Eye Irritation

Primary skin and eye irritation testA were carried out accordingto the modified Draize method as described in the Code of Federal Regulations../Rabbits were clipped free of hair over the appropriate skin areas at least24 hr prior to testing. After application of the test material, as a 50%paste with peanut oil, the irritation score on intact and abraded skin andthe eye was evaluated at 24 and 72 hr.

E. Dermal Sensitivity Studies

Dermal sensitivity test was performed in guinea pigs accordingto the "maximization test" described by Magnusson and Kligman.i! Testanimals were clipped free of hair at least 24 hr prior to testing or

prior to the final challenge with the test substances. The preparationsof the nitrotoluenes used for the dermal sensitization tests were thesame as those used for acute oral toxicity study.

F. Disposition and Metabol.sm

I. Radiochemicals

The 1 4 C-labeled 3,5-DNT and 4-ADNT (ring-UL- 1 4 CO were preparedby the New England Nuclear Corporation (Boston, Massachusetts). Theirspecific activities were 2.29 and 4.06 mCi/mM, respectively. They wereradiopure by thin-layer chromatography.

2. Experimental Procedures

Charles River CE® female rats wei3hing between 175 and 250 gwere used. Each rat was fasted overnight before being given a singleoral dose of approximately 1/10 of the LD50 of the test compound, spikedwith 10 pCi of the 1 4C-labeled compound. The test material was suspendedin peanut oil and given via an intragastric tube at a volume of 1 ml/lO0g body weight. After dosing, each rat was placed immediately in a "Roth-Delmar" metabolism cage29! with food and water ad libitum. The chamber was

4 .

vented continuously with C02-free air at a rate of M50 ml/mmn. Expired CO2

was collected by bubbling the air through six absorption columns connectedin series. Each column contained 100 ml of 5% NaOH. Feces and urine werecollected separately in the apparatus. At the termination of each experi-

.s ment, the rat was anesthetized with ether and aortic blood collected in aheparinized syringe. Liver, kidneys, brain, lungs, and thigh muscle wereremoved, weighed, and representative samtles taken for analysis of radio-activity. The gastrointestinal tract plus contents (GI) was removed andweighed. The GI and the feces were homogenized in three volumes of dioxaneim0/and filtered; samples of the Ziltrate and the filtered residue were assayedfor radioactivity.

3. Radioactive Assays

Aliquots of whole blood, tissue samples, and filtrate residueswere digested in 2N NaOH. The carcasses were digested in 10 volumes of6N NaOH. Blood samples were decolorized by dropwise addition of hydrogenperoxide. Sawples of tissue digests were neutralized with Beckman BBS-2,

solubilized Ja Beckman BBS-3, and counted in a toluene-PPO-dimethyl POPOPcocktail using a Packard Tricarb 3375 liquid scintillation spectrometer.Samples of plasma, urine, dioxane filtrates, and test radiochemicals weresolubilized directly in BBS-3 and counted. 1 4C02 samples from the airtraps were spotted on filter paper, dried, Pnd counted. All data were cor-rected for background and quenching.

4. Thin-Layer Chromatography (TLC)

Precoated Silica Gel 60 plates (without fluorescent indicator,0.25 mm thickness, E. M. Laboratories, Inc., Elmsford, New York) were usedfor all experiments. All samples were spotted 2.0 cm frote the bottom ofthe plate and developed for a minimum of 10 cm. The solvents used were:(a) n-butanol:methanol:water (120:33:57, v/v/v); (b) petroleum ether:ethyl acetate (15:85); and (c) 1,2-dichloroethane:petroleum ether (25:75).Nitrotoluenes were detected using 5% diphenylamine spray reagenlt/ fol-

lowed by UV-irradiation.

G. Ames Test

1. Preparation of Test Cc".iounds

All nitrotoluenes, nitroglycerins, and TNM were dissolved in di-

methylsulfoxide at concentrations varying from 1 to 10 mg/ml. WP was pre-pared from a saturated solution in distilled water ("phossy water"). NCwas prepared as a suspension of 1 to 5 mg/ml in distilled water.

5

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2. Bacteria and Culture Media

The bacteria used in our experiments were the Salmonella typhi-murum tester strains TA-17.5, TA-1537, TA-1538, TA-98, and TA-l00. Thesetester strains are histidine auxotrophs and are used to detect base-pairsubstitutions (TA-100 and TA-1535) and frame-shift reverse mutations(TA-98, TA-1537, and TA-1538), All tester strains were obtained fromDr. Bruce Ames, University of California, Berkeley.

The selective medium for the histidine auxotrophs in the mu-tagenesis assays was Minimal Agar-Davis (Difco, Detroit, Michigan). Difconutrient broth was used to prepare stock cultures.

3. Preparation of Rat Liver S-9 Fraction

The S-9 fraction from rat liver was prepared according to theprocedure of Ames et al.2-a/ Briefly, male Charles River CD rats weighing180 to 200 g were given intraperitoneal injections of sodium phenobarbitalat a dose of 80 gm/kg of body weight for four consecutive days. Twenty-fourhours after the last dose the rats were killed. The livers were asepticallyrcmoved, washed once in 10.0 ml of ice-cold, sterile 0.15 H KCl, put into3.0 ml of ice-cold, sterile 0.15 H KCl/gm of liver. The liver was thenminced with a sterile scalpel and scissors, transferred into a cold Teflonin glass homogenizer and homogenized (25% w/v) at low spedd (three to fivestrokes). The homogenates were transferred into sterile centrifuge tubesand centrifuged for 10 min at 9,000 x g. Tne supernatant (S-9 fraction)was decanted; 2-ml aliquots were frozen in an ethanol-dry ice bath andstored at -80*C in a Revco freezer.

Prior to use, each batch of S-9 fraction was tested for its effi-ciency for metabolic activation. For these tests, the TA-100 and theTA-1535 strains were used with 7,12-dimethylbenzanthracene (20 ug/plate)and cyclophosphamide (200 ug/plate) as the reference mutagens, respectively.Increasing amounts of S-9 fraction were added to complete the test systemand the cultures incubated as in the normal assay. Each assay was runagainst a batch of S-9 fraction previously determined to give satisfactoryresults with the test chemicals. The concentration of the S-9 fractionfrom the new batch was then adjusted to give results comparable to thereference batch.

4. Microsomal Activation System

The microsomal activation system used for the mutagenic assaycontained per milliliter: S-9 fraction (0.08 ml), NADP (4 OM), glucose-6-phosphate (5 jM), KCl (33 uM), AL1C]-) (8 jaM), and sodium phosphate buffer(100 uM), pH 7.4. The microsomal activation system was prepared freshdaily.

6

L ...... d

5. Assay Procedure

The histidine-requiring Salmonella typhimurium tester ntrainsr TA-1535, TA-1537, TA-1538, TA-98, and TA-1O0 were exposed to selected con-

centrations of each test compound. The standard concentrations were 1,000,300, 100, and 10 og/plate (0.1 ml) to insure a wide dosage range. For[i those compounds showing bactericidal activity, as indicated by a reductionin the number of spontaneous revertants in the treated plates relative tothe ccntrol, additional concentrations of 1.0 to 0.01 ug/plate were used.For NC, the highest concentration used was 5,000 og/plate. For WP, a

.I saturated aqueous solution was then serially diluted to 1/2, 1/10, 1/33,and 1/100. All compounds were prepared for testing as described above andrun in duplicate with and without metabolic activation. 7,12-Dimethyl-benzanthracene (20 pg), benzo(alpyrene (5 ug), and cyclophosphamide (200ug) were used as positive controls to assure that all strains were capableof mutation during a particular experiment, and that the metabolic activationsystem was working properly. All cultures were incubated for 48 hr at 37*Cand the number of revertant colonies on each plate counted. The mutagenassay was scored as the ratio of the number of colonies (total revertants)in the experimental plates over the number of colonies in the control plates(spontaneous revertants). This was taken as the mutagenic ratio. A com-

pound was considered to be mutagenic if its mutagenic ration was > 2.0.1a/Our criterion for a strong mutagen was a compound having a mutagenic ratio

O 2.0 at concentrations less than 100 ug/plate. A weak mutagen requiresgreater th.Jn 100 g/plate to produce a mutagenic ratio > 2.0.

17[

[7

I

I, 111. RESULTS

L :A. Synthesis of 3.5-DNT

1. Identity

L. a. Capiyl eltinr Point

Observed - 91 to 92CReported5/ - 93-C

.i b. Infrared SpectrumFThe 'Infrared (IR) spectrum (KBr wafer) of prepared 3,5-DNT1was consistent with that rc±pored.12/

c. Nuclear Magnetic Resonance

The NMR spectrum in deuterated chloroform of p-repared"3,5-DNT was consistent with that reported._3/

2. Assay

a. Gas Chromatography (GC)

The prepared 3,5-DNT was studied using the following system:

Instrument: Varian 200Detector: Flame ionizationColumn: 6 ft x 18/ in., glass *

1.5% GEXE-601.5% DC-LSX-3-0295 on Gas Chrom Q

Colunn temperature: 170CInjector temperature: 175CDetector temperature: 200oC

I Nitrogen flow: 40 cc/min

By peak area comparison, the prepared sample contai.ns 98%3,5-DNT and 2% of an unknown component with a retention time of 1.7 rela-tive to that of the 3,5-DNT.

9 ii mommaa

[i~

II

b. Thin-Layer Chromatography (TLC)

1. Plate: Brinkman Silica Gel F

2. Solvent system: Ethyl acetate:petroleum ether (15:85)

3. Material spotted: 100, 10, 5, 2, and 1 ug sample4. Detection: UV, 254 nm

The prepared sample showed a major spot at Rf 0.69 and aminor spot at Rf 0.77. Based on spot intensity, the minor spot has a con-centration of 2%.

c. Discussion

TLC and GC results indicate the sample to be 98% pure with2% of an organic impurity. Based on the synthesis method and sample workup

procedures, the impurity may be an isomeric nitrotoluene.

B. In Vivo Studies

The acute oral LD5 0 s of all nitrotoluene derivatives tested, in-

cluding those previously reported,!/ in rats and mice are listed in Tables 1and 2, respectively. The results of the primary skin and eye irritation

tests in rabbits are shown in Tables 3 and 4. The results of the dermalsensitivity tests in guinea pigs are listed in Table 5.

1. 3,5-Dinitrotoluene

The acute LD5 0 ± S.E. (95% confidence limits) of 3,5-DNT in maleand female rats were 309 + 13 (278-340) and 216 + 19 (160.-256) mg/kg,respectively (Table 1). In male and female mice, they were 611 + 43 (523-714) and 607 + 21 (559-650) mg/kg, respectively (Table 2). No toxic signsexcept ataxia of scme animals given the higher doses were noted.

3,5-DNT was non-irritating to rabbit skin and eyes and was not asensitizing agent to guinea pigs (Tables 3, 4 and 5).

Table 6 summarizes the recovery of radioactivity after a single

oral dose of 3,5-DNT (ring-UL- 1 4 C). The radioactivity remaining in the GItract averaged 65.0% and 2.9% of the administered dose at the end of 4 and24 hr, respectively. The recovery of radioactivity in the feces during thesame time periods were 0.9% and 21.7%. From these values, we estimate thatat least 75% of the dose was absorbed and that the absorption was essentiallycomplete at 24 hr. Host of the absorbed radioactivity was excreted in theurine, averaging 77.2% of the administered dose at 24 hr. No radioactivitywas recovered 4n the expired air. Only small amounts of radioactivity werefound in the various tissues. At 4 hr, the radioactivity ratios in the

10

tissues, relative to plasma, were in the following order: kidneys >I liver " spleen, lungs > muscle, brain. At 24 hr, the radioactivity ratiosof liver and muscle were approximately twice that at 4 hr, whereas the radio-activity ratios of the other tissues remained about the same.

The TLC analysis of 24-hr urine from 3,5-DNG (ring-UL-1 4 C)-treatedrats using ethyl acetate:petroleum ether and butanol:taethanol:water solventsystems demonstrated a single radioactive band which did not correspond to

the parent compound (Figure 1). This radioactive component(s) had an Rfvalue of 0.65 with the butanol:methanol:water system.

2. 2-Amino-4,5-Dinitrotoluene

The acute LD5 0 + S.E. (95% confidence limits) of 2-ADNI in male

and femile rats were 2,240 + 85 (2,070-2,430) and 1,394 + 191 (989-2,830)

mg/kg, respectively (Table 1). In male and female mice, they were 1,722 +154 (1,450-2,131) and 1,522 + 71 (1,372-1,692) mg/kg, respectively (Table

2). In both species, the toxic signs were inactivity, depression, uncon-sclousness and slow, gasping breathing. In lethal doses, symptoms wereobvious within 30 min, and most deaths occucred 1 to 24 hr after dosing.

Although 2-ADNT is bright yellow, the surviving rats and mice excreted orangeurine for a few days after dosing. Rats, but not mice, who survived theinitial depression were hyperactive for several days. Their gait was hopping;

they appeared on the verge of convulsing, although when disturbed they would

jump several inches in the air rather than convelse. They appeared extremelyunkempt, with urine-stained, bristly hair. Most of these rats returned tonormal in 7 to 10 days, but some rats and some %ice died as long as 10

days after dosing.

2-ADNT was a mild skin irritant (Table 3), but non-irritating tothe rabbits' eyes (Table 4). It was not a sensitizing agent in guinea pigs(Table 5).

3. 4-Amino-2,6-Dinitrotoluene

The acute LDso ± S.E. (95% confidence limits) of 4-ADNT in maleand female rats were 1,360 + 53 (1,W60-1,465) and 959 + 76 (787-1,154)mg/kg, respectively (Table 1). In male and female mice, they were 1,342 +

107 (1,141-1,611) and 1,495 + 90 (1,318-1,713) mg/kg, respectively. Towicsigns included inactivity, depression, unconsciousness, and slow, gaspi',Igbreathing, and appeared within 30 min of dosing. Most deaths occurred 1 to24 hr after dosing. However, some animals recovered from the depresson

within a day, then died as long as 10 days after dosing. Mice dou.. withthe orange 4-ADNT excreted reddish-orange urine, which stained their hairfor a few days. Rats recovering from the initial depression were hyperac-tive, with a hopping gait and extremely unkempt appearance, for several days.

L 11

--i

4-ADNT was not irritating to rabbits' skins and eyes and wasnot a sensitizing agent in the guinea pigs (Tables 3, 4 and 5).

Table 7 summarizes the recovery of radioactivity after a singleoral dose of 4-ADNT (ring-UL- 1 4C). The radioactivity remaining in the GItract averaged 70.1% and 5.5% of the administered dose at the end of 4 andA24 hr, respectively. The recovery of radioactivity in the feces duringthese same time periods were 0.6% adn 44.4%. From these values, we estimate

[ that at least 50% of the dose was absorbed and that the absorption was es-sentially complete at 24 hr. Most of the absorbed radioactivity was ex-.reted in the urine, averaging 30.1% of the administered dose at 24 hr.A negligible amount (0.2%) of the radioactivity was recovered in the expiredair. Only small amounts of radioactivity were found in the various tissues.At 4 hr, the radioactivity ratios in the tissues, relative to plasma, werein the following order: kidneys > liver > spleen > lungs > brain > muscle.At 24 hr, the radioactivity ratio of the liver was three times that at4 hr; the ratios of kidney and lung doubled, whereas the radioactivityratios of the other tissues remained about the same.

TLC analysis of 24-hr urine from 4-ADNT (ring-UL-1 4 C)-treatedrats using ethyl acetate:petroleum ether and butanol:methanol:water sol-vent systems demonstrated two radioactive components, neither of whichcorresponded to the parent compound (Figure 2). Both components migratedwith the butanol:methanol:water solvent system. The first, more polar com-ponent had an Rf value of 0.57 and represented about 78% of the total ra-dioactivity in the urine. The other component had an Rf value of 0.87 andrepresented 22% of the total radioactivity in the urine.

C. Ames Tests

The mutagenic activity of the munitions compounds tested is pre-sented in Table 8 as mutagenic ratios. Table 9 summarizes the criticalvalues. Four compounds including 1,3-DNG, 1-MNG, NC and WP were not mu-tagenic in the test system. The other 11 compounds displayed various de-grees of mutagenic activity.

Five compounds including TNT, 2,4-DNT, 2,5-DNT, TNM, and 1,2-DNG,

exhibited significant increases in the number of revertants at 10 or 30pg/plate. With the exception of 1,2-DNG, these compounds produced bothbase-pair substitution and frame-shift mutations. 1,2-DNG produced onlyframe-shift mutation as indicated by the reversion of the TA-1537 strain.TNT appeared to be the most potent of the compounds tested. As little as10 ug/plate of TNT was mutagenic in the TA-98, TA-1538 and TA-1537 strains.

12

1lh f When 30 pg/plate were used, TNT was positive in four test strains; at 300Iu •8g/plate, TNT was positive in all five tester strains. 2,5-DNT shoved posi-

tive response in all five tester strains but at a higher concentration thanTNT. TN14 was bactericidal at concentrations above 1.0 ug/plate without ac-tivation; however, S-9 fraction reduced the toxicity and produced a mu-tagenic response in strains TA-1535 and TA-98. On the other hand, the

! 1 utagenic activity of 2,4-DNT was decreased and that of 1,2-DNG was com-pletely inactivated in the presence of S-9 microsomal fraction. 1,2-DNGwas mutagenic in the TA-1537 strain without activation.

Six compounds including 2,3-DNT, 2,6-DNT, 3,4-DNT, 3,5-DNT, TNG,and 2-MlG displayed a weak mutagenic activity in these test systems.These compounds required between 100 and 1,000 ag/plate to produce a signifi-

Scant increase in mutation frequency with or without metabolic activation.3,4-DNT and 2-MNG produced only base-pair substitution mutation; 2,3-DNT

* I and 2,6-DNT produced only frame-shift mutations; 3,5-DNT and TNG producedboth base-pair substitution and frame-shift mutations. 2,3-DNT and 2-INGrequired metabolic activation. On the other hand, the mutagenic activityof 3,5-DNT was decreased and that of 2,6-DNT was completely inactivated inthe presence of the S-9 microsomal fraction.

Ii

13

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15

PRIMARY SKIN ,UITATIoN OFVARIOUS NICROaEZCOMPOUNDS IV RA"BITS

TNT 1.6b/

,3-DNT 1.782,4-DNT 0.25

2,5-DNT 3.80-2 ,6-DNT 0.2 1d,3,4-DNT 2. 003,5-DNT < 0.22 -ADNT 0.21

4-ADNT < 0.2Peanut Oil.

(vehicle control) 0.33

a/ Average value of six rabbits with intac.t .ad abraded skin in eachtest group. The compounds are classified as follows:

> 0.2 over controls is uild irritant

> 2.5 over controls is moderate irritant> 5.0 over controls is severe irritant

b/ Red color under all patches at 24 hours.C/ No edema was apparent but the entire area covered by the compound was

undergoing necrosis in 24 hours in both the intact and abraded skin.

di Yellow coior under all patches at 24 hours.

1.616

SI.12. .TABU 4

Twrb/Noirtn2 3Nonirritant

NonirritantS2 .4-1W1•

~Nonirritont

-Nonirritant

#Nonirrltant2 AM

Nonirritant4-ADWzf

Nonirritant

Peanut Oil konlrrytant('fQhtce Itcontrol)

":E/ Si rabbits Per teot groap,b/ Red color around the eya at 24 hours.S/ Yellov color around the eye at 24 hours,

Ji

i 17

TABLE 5

DEI&L IWUITIVMRY UL? Y mk1Oa SaLTOUm=C

TNT 4/10 40% ett

2*3-ONT 0/10 0 mn

2 *4-DtT 0/10 0 nomo

2$5*DNT 0/10 0 come

2$6wDNT 2/10 20% slid

34OT0/10 0 *ODA

35DT0/10 0

4-ADW 01

TABIX 6

% of Admaimstered pose flBadioactivity Batio-6/

4 Hour 24 Hour 4 Hour 24 Hour

Gastrointestinal tractplus contents 65.0 + 6.3!I 2.9 + 1.0

Faces 0.9 + 0.3 21.7 ± 3.7Whole Blood!/ 1.1 ± 0.3 0.1 + 0.0Expired Air eD0 /Urine 24.6 + 3.3 77.2 + 2.2Spleen 0.1 + 0.0 < 0.1 0.6 + 0.1 0.6 + O.1SLver 0.8 T 0.2 0.1 + 0.0 1.0 ± 0.2 2.5 ± 0.1Kidneys 0.5 T 0.1 < 0.1 2.6 T 0.3 2.9 ± 0.4Brain 0.1 + 0.0 < 0.1 0.3 T 0.0 0.4 T 0.0Luna b/ 0.1 + 0.0 < 0.1 0.6 + 0.0 0.6 + 0.1Skeletal Muscle- 2.9 T 0.6 0.3 + 0.0 0.3 ± 0.0 0.6 ± 0.1

Recovery 96.6 + 2.7 103.8 + 2.2

a/ Based on 7% of the body weight.b/ Based on 40% of the body weight.S/M Nan + S.I. of three rats.d/ Mean of two rats.e/ Radioactivity in 1 ml or gm of wet tissue per radioactivity in 1 ml of plasma.Note: ND - Not determined

19------------

TABLE 7

DISTRIBUTIONI AND EXCRETION OF RADIOACTIVITY IN RATS-RECEIVING

4-!ANO-2.6 -DINTMOLUWEN- (RING-UL- 14C)

% of Administered Dose Radioactivity Ratio4 Hours 24 Hours 4 Hours 24 Hours

GI and contents 70.1 + 4.12 5.5 + 1.1Feces 0.6 ± 0.1 44.4 T 12.4Whole blood'/ 0.6 ± 0.2 0.2 + 0.0Air ND 0.2 + 0.1Urine 11.1 + 3.5 30.1 + 9.5Spleen < .1 < .1 1.4 + 0.6 1.2 + 0.1Liver 0.9 + 0.2 0.5 + 0.1 2.6 + 0.5 8.2 + 0.6Kidneys 0.4 + 0.1 0.1 + 0.0 4.8 + 0.7 7.7 + 0.4Brain 0.1 + 0.0 < 0.1 0.9 + 0.1 0.8 + 0.2Lungs b/ 0.1 + 0.0 < 0.1 1.3 + 0.2 2.0 + 0.2muscle-b/ 2.4 + 0.6 0.3 + 0.0 0.6 + 0.1 0.6 + 0.1

Recovery 86.4 + 8.1 81.2 + 4.5

a/ Based on 7% of the body weight.b/ Based on 407. of the body weight.c/ Mean + S.E. of three rats.d/ Radioactivity in 1 ml or gm of wet tissue per radioactivizy in 1 ml of

plasma.Note: ND - Not determined.

I

20 b-

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60

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*40

20

< 00.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

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F 60

B 35 -DNT*40-

i200-0

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00! Rf

Figure 1 - TLC of Rat Urine After Oral Administrationof 3,5-DNT (Ring-UL- 14 C)

25

24 Hr. UrineSolvent (Ethyl Acetate: Petroleum Ether)

100 -4-ADNT

50 !"50

0 0 0.2 0.4 0.6 0.8 1.0

RF

24 Hr. UrineSolvent (BuOH: MeOH: H20) 1

50 j

25-4-ADNT

010 0.2 0.4 0.6 0.8 1.0 ,

Rf

Figure 2 - TLC of Rat Urine After Oral Administration -

of 4-ADNT (Ring-UL- 1 4 C)

26 :11 ''~ ~Mt,.

IV. DISCUSSION

A. Acute Oral Toxicity in Rats

In male and female rats, 3,5-DNT was the most toxic nitrotoluenecompound (LD5 0 s of 309 and 216 mg/kg, respectively), 2-ADNT was the least

r toxic nitrotoluene (LD50s of 2,240 and 1,394 mg/kg, respectively) and 4-ADNTwas the second least toxic nitrotoluene (LD 5 0 s of 1,360 and 959 mg/kg, re-spectively). The two ADNTs were distinctly less toxic than TNT itself, while2,3-DNT and 3,4-DNT, the two orthodinitro isomers, had toxicity similar toTNT. In the other nitrotoluenes, the two nitro groups are meta or para toeach other, and toxicity was intermediate between that of TNT and that of3,5-DNT.

B. Acute Oral .:oxicity in Mice

As with the rats, 3,5-DNT was the most toxic nitrotoluene com-

pound tested with LD5 0 s in male and female mice of 611. and 607 mg/kg,respectively. In male mice, 2-ADNT (LD5 0 of 1,722 mg/kg) and 4-ADNT(1,342 mg/kg) were t1,F second and fourth least toxic, in female mice theywere the two least ._xic (LD5 os of 1,552 and 1,495 mg/kg, respectively).Although the order oi toxicity varies, 2,3-DNT, 2,4-DNT and the ADNTs areless toxic than TNT, and the other isomers are more toxic, in both sexes.

C. Toxic Effects

As with the nitrotoluenes tested earlier, the toxic effects seemedto be on the central ervous system: inactivity, unconsciousness and (inrats given ADNTs) a later hyperexcitability and failure to groom. Mostdeaths occurred wiV t 24 hr of dosing, but the later deaths in rats andmice and the longer lasting effects in cats suggest the presence of twotoxic mechanisms: first, a direct depression leading tu unconsciousnesswhich deepened into respiratory paralysis; secondly, a slower, less welldefined action produc 4 ng the motor excitability and failure to groom. This

latter phase may be related to the convulsions seen with some nitrotoluenecompounds, such as TNT and 3,4-DNT.!/

Mice given either 2-ADNT or 4-ADNT and rats given 2-ADNT pro-duced urine of a redder color than the administered compound. This redshift suggests that the metabolism of the compounds affected the substitu-

ents on the aromatic ring, probably reducing the nitro groups to amines,this changing the chromophore.

II 27

L _.. . ..

D. Primary Skin and Eye Irritation

All three compounds tested here (3,5-DNT and the ADNTs) producedless irritation to intact and abraded rabbit skin than the vehicle control.All three were non-irritating to rabbit eyes. These results are similar tothose of the other compounds tested,

E. Dermal Sensitivity

None of these three compounds produced dermal sensitization in

guinea pigs.

F. Disposition and Metabolism

Qualitatively, the disposition and metabolism of 3,5-DNT and4-ADNT were much like those reported earlier.-/ The compounds were wellabsorbed in 24 hr after oral dosing. The extent of absorption of 3,5-DNTwas similar to that of TNT. 4-ADNT was less well absorbed but was similarto 2,3-DNT and 2,6-DNT. Both 3,5-DNT and 4-ADNT were widely distributedin various tissues and slightly concentrated by the liver and kidneys.

A majority of the administered 3,5-DNT appeared in the urine in

24 hr, but none of it chromatographed as 3,5-Drt itself. More 4-ADNT-derived radioactivity appeared in the feces than in the urine; the radio-activity in the urine apparently included no 4-ADNT. Therefore, both com-

pounds were metabolized extensively in the body. The aromatic ring apparently

remained intact; negligible radioactivity appeared in the expired air.

G. Ames Tests

The munitions compounds studied in this investigation are eithernitroaromatics or nitroaliphatics, with the exception of white phosphorus.All of the nitroaromatics studied appeared to be mutagenic to Salmonellatyphimurium. Many recent studies have shown that, in general, nitroaro-matics exhibit a high degree of mutagenic activity in the Salmonella/microsome plate test.14"18/ These nitroaromatics are thought to exhibit ahigh degree of mutagen activity to microbial test systems because of theiractivation by nitroreductase(s) and other metabolizing enzyme,15/ endogenousto the microbial tester strains. Furthermore, liver homogenates have re-cently been shown to activate nitroaromatic compounds to mutagens. 1 9 /In light of these considerations, it appears that TNT and the six DNT isomersshould be considered as potentially mutagenic and possibly carcinogenic.

281!'

The other class of munitions compounds showing mutagenic activitywere the nitroglycerins. TNG is metabolized by mammals to inorganic ni-trite. 2 0,21/ Hodgson et al.2_2/ have recently reported that the DNGs andMNGs are also metabolized by denitration. It has been shown that inorganicnitrite will react with endogenous primary and secondary amines to formhighly mutagenic and carcinogeuic nitrosamines.23/ Since the major metabo-

lite of the nitroglycerins is inorganic nitrite, this class of compounds

should also be considered as potential mutagens and/or carcinogens.

[ The results of this investigation suggest that several commonI. munitions compounds and chemical intermediates are mutagenic. The tester

strains used in this study are currently used in many laboratories as aprimary screen of compounds for potential carcinogenic activity. However,

the limitations of the Salmonella/microsome plate test should be fully un-derstood before any conclusions can be made on these compounds. A compounddemonstrating a positive response in the Salmonella/microsome plate test

I. may not, in fact, be carcinogenic or even mutagenic in mammalian systems.The Salmonella typhimurium tester strains used in this study have been re-ported to be unusually sensitive for the detection of nitro-containing

I. mutagens and carcinogens.-6/ Since many of the compounds did not require

metabolic activation or were inactivated in the presence of S-9 microsomalI fraction, it is quite possible that the bacteria may contain nitroreductase(s)

not found in mammals which result in a false positive in the Salmonella/microsome plate test. Two of the compounds, 2,4-DNT and TNG, displayingmutagenic activity in this study are currently being tested for carcinogenic

activity in animals and preliminary findings suggest that 2,4-DNT is car-cinogenic to rats. 2 4 /

Iiiii

Ii

[ 291! i

V. CONCLUSIONS

The e.cute oral toxicities of all the nitrotoluenes tested aregenerally similar. 3,5-DNT is the most toxic (LD5Os from 216-611 mg/kg)and 2-ADNT the least toxic (LD5 0o from 1,394-2,240 mg/kg). Acute toxiceffects are primarily on the central nervous system: depression, sometimeswith hyperreflexia or convulsions. After some compounds, the animals ex-creted brightly colored urine, of the color of the compound or of a longerwavelength. Only 2,5-DNT was a moderate skin irritant; the others were non-irritating to rabbit eyes. TNT was moderately sensitizing to guinea pigs,2,6-DNT mildly sensitizing, the others nonsensitizing.

Al] these nitrotoluenes were fairly well absorbed and widelydistributed by rats. They were concentrated in the liver and kidneys.They were extensively metabolized, preaumably in the liver, and excretedin the urine.

Ames tests of various munitions compounds found that TNM, TNT,2,4-DNT, 2,5-DNT and 1,2-DNG were potential mutagens, active at 10 to 30tpg/plate. The other nitrotoluenes tested (2,3-MbN£, 2,6-DNT, 3,4-DNT and3,5-DNT) and TNG and 2-.-MNG were weakly mutagenic. 1,3-DNG, I-MNG, NCand WP were not mutagenic in this test.

31A: ] •31

II VI. CORRIGENDA TO REPORT NO. 1

The following typographical errors have been noted in ProgressReport No. l,1/ and should be corrected.

Page vii, line 3: The parenthetical phrase should read: (1,2-DNG and 1,3-

DNG).

Page 3, line 19: Section "b" heading should read: 1,3-DNG:

Page 15, line 28: The reference should be to Figure 7.

Page 31, Table 2: The LD5 0 for females for 1,2-DNG, should have no decimalpoint, and should read: 1,423 + 176.

3

~.E

I

I 33 ____________..... ________

.1_ _

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9. Roth, L. 3., E. Leiter, J. R. Hogness, and W. r. Langham, Studieson the Metabolism of Radioactive Nicotinic Acid and Nicotinamidein Mice, J. Biol. Chem., 176:249-257, 1948.

35

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36 1-n ~ . .- . -<~--.. -'-- . . a -- . t,]. .

lI

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22. Hodgson, J. R., J. P. Glennon, J. C. Dacre, and C. C. Lee, Metabolismand Disposition of Isomers of Dinitro- and Mononitroglycerol in theRat, Toxicol. Appi. Pharmacol., 40:65-70, 1977.

23. Sander, J., F. Schweinberg and H. P. Menz, Untersuchungen ueber die

Entstehung Cancerogenes Nitrosamine im Magen, Hoppe-Sylers Z.Physiol. Chem., 349:1691-1697, 1968.[

24. Ellis, H. V., III, C. B. Hong, J. C. Dacre, and C. C6 Lee, Chronic

Toxicity of 2,4-Dinitrotoluene in the Rat, Toxicol. A1,ij Pharmacol.,[ 45:245-246, 1978.

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